As axons pathfind to their targets in the developing nervous system, they are guided by a complex environment of positive and negative cues, which are sensed and transduced by the machinery of the growth molecules involved in pathfinding, but many remain unknown. A large- scale genetic screen in zebrafish has isolated approximately -25 genes required for the projection of retinal axons to the tectum. Of these, the ashtray mutant shows the most severe and specific pathfinding phenotype. Retinal axons in ashtray seem to ignore many guidance cues: they cross the midline repeatedly, and project anteriorly to forebrain and posteriorly to hindbrain as well as to their normal target. There is no general derangement of brain patterning, and the early axon scaffold is undisturbed. Embryonic eye transplants show that ashtray acts eye- autonomously. Thus, the ashtray gene product is likely to be an axon pathfinding molecule required in retinal axons. We have genetically mapped ashtray at high resolution, and found it maps very close to a novel zebrafish roundabout homolog, zRobo1A. The experiments proposed here are designed to understand how ashtray acts during retinal axon pathfinding and to elucidate the molecular nature of the gene. First, axons will be labeled in fixed embryos and growing axons observed in live embryos to determine when and where ashtray axons misroute. Cell type-specific markers will also be used to check whether ashtray acts to change retinal ganglion cell fate. Second, single- cell ashtray is required at distinct choice points during pathfinding. Third, six specific sets of non-retinal axons that normally exhibit a wide variety of behaviors will be labeled to test whether they are affected in ashtray. Fourth, the ashtray gene will be cloned. We will first test whether it is a defect in zRobo1A. If not, we have begun a genomic walk and will clone the gene positionally. In summary, this project will advance our understanding of how wildtype retinal axons pathfind; illuminate where, when, and how the behavior of single zebrafish retinal axons requires the function of ashtray, be established for the future analysis of all the zebrafish retinotectal mutants, which promise to yield important insights into the genetic control of visual system development.